Emerging evidence supports the proposition that the mitochondrial respiratory chain (MRC) functions via organized multicomplex structures called supercomplexes. However the dynamics and regulation of supercomplex assembly have not been fully investigated. In particular, hardly any regulatory protein factors involved in supercomplex assembly have been identified. Our long term goal is to understand the dynamics of mitochondrial respiratory machinery and its underling regulatory mechanism. The objective of this particular application is to determine if the mitochondrial chaperon, 75 kDa glucose regulated protein (Grp75) plays a role in regulating supercomplex assembly and further to identify additional protein factors involved in this important process. The study of mammalian respiratory supercomplex assembly has been difficult since common yeast systems, which could be utilized as a powerful genetics system to identify putative regulatory factors, lack Complex I an essential component of mammalian supercomplexes. We have previously established an efficient method to isolate cells carrying mitochondrial DNA (mtDNA) mutations and further generated several cell models with regulated/altered supercomplex assembly, probably due to the enhanced/stabilized interactions between supercomplexes and regulative factor(s). Characterizations of these cell lines employing both molecular and proteomics approaches have implicated the molecular chaperone Grp75 supercomplex assembly. Interesting Grp 75 has previous been implicated in Parkinson's diseases (PD) due to 1). Grp 75 mutations have been identified in PD patients; 2) Low Grp 75 expression was found in brains of PD patients; 3). Our preliminary studies showed heterozygous Grp75 mice exhibited lower motor activities associated with defective supercomplex assembly. The central hypothesis for this application is that Grp75 is an essential part of machinery which regulates the assembly of supercomplexes, and defective of supercomplex assembly associated with deficient Grp 75 would lead to neuro-degeneration. To test this hypothesis, we propose to pursue the following three specific aims: 1) Characterize the role of Grp75 in supercomplex assembly. In particular, we will follow the step-wise assembly and degradation of individual complexes and supercomplexes in presence and absence of Grp75 with newly developed approaches in the lab; 2) Determine the regulatory mechanisms of Grp75 on supercomplex by Identify novel protein factors involved in regulating supercomplex assembly. With proteomic analysis of proteins interacting with Grp75 and Complex I containing supercomplexes in the cell models with regulated/altered supercomplex assembly, we aim to isolate novel protein factors involved in regulating supercomplex assembly. 3) Characterize the mouse models with altered expression of Grp75. The implications of defective supercomplex dynamics in neuronal degeneration will be further explored in heterozygous and neuronal-specific Grp75 knockout mouse models. We will investigate the underlying molecular pathways derived from Grp75 defect to supercomplex deficiency to neuronal degeneration. The approach is innovative, because it combines our unique cell models exhibiting upregulated supercomplex dynamics with newly-developed analytical methods to allow understanding of the complexity of respiratory supercomplex assembly. The establishment of novel mouse models with defective supercomplex dynamics should open new possibilities to study bioenergetics in neuronal system and neuro-degeneration. We believe that we are in a strong position to characterize respiratory supercomplex assembly. The research is significant, because elucidating this mechanism could provide new insights into the regulation of oxidative phosphorylation machinery. In addition, we anticipate our work will also help to identify novel risk genes involved in neurodegenerative diseases associated with mitochondrial dysfunction.